Tutorial: Cryo_fit2: Fit Biomolecules into Cryo-EM Maps using dynamics (commandline)

Contents

Note

cryo_fit2 is under active development.

Therefore, consider to use cryo_fit1 instead (especially for RNA modeling).

Overview

This tutorial will show you how to fit biomolecule atomic structures into cryo-EM maps using dynamics simulation with PHENIX commandline

Theory for cryo_fit2

See the theory notes for cryo_fit2

Input

  • The <initial_model> is a guide or template structure (.cif/mmCIF/.pdb) that is close to a <target_map> (.ccp4/.map) structurally.
  • A user can use map_to_model to build the atomic model.
  • A user can use either dock_in_map or UCSF chimera (Tools->Volume Data->Fit in Map) to roughly place the model into a map.
  • The <target_map> should have decent density.
  • Most mrc type cryo-EM maps have decent density.
  • However, some maps (like from UCSF Chimera's map segmentation) have low density. Therefore, cryo_fit2 will have just little incentive to fit to it.

  • For example, left yellow maps ares fine. However, right gray/white maps lack enough density to run.

    ../images/cryo_fit2_example_map.jpg ../images/cryo_fit2_example_map2.jpg
  • To fit into these low density maps, Doo Nam tried very high map_weights (like 10^8). However, these efforts either broke atomistic model geometries or did not budge the atomistic model.

How to run

At command line:

% phenix.cryo_fit2 <initial_model> <target_map> <map resolution in Angstrom>

For example:

% phenix.cryo_fit2 model.pdb map.ccp4 3.5

Tutorial command:

% Go to <user phenix>/modules/cryo_fit2/tutorial

% source run_me.sh

%% (run_me.sh is phenix.cryo_fit2 input/tutorial_cryo_fit2_model.pdb input/tutorial_cryo_fit2_map.ccp4 resolution=4)

How to add user specified geometries

  1. If a user specified geometry file is small, then simply copy/modify <user phenix>/modules/cryo_fit2/tutorial/eff_file_template/user_custom_geom.eff, and modify it for a user's need.

  2. If a user specified geometry file is large, then run:

    % phenix.cryo_fit2 <initial_model> <target_map> <map resolution in Angstrom> <write_custom_geom_only=True> <HE_angle_sigma_scale> <HE_sigma> <parallelity_sigma>

For example:

% phenix.cryo_fit2 model.pdb map.ccp4 3.5 write_custom_geom_only=True HE_angle_sigma_scale=0.04 HE_sigma=0.2 parallelity_sigma=0.02

Then, automatically generated geometry file should be generated at the same folder.

Modify it for a user's need.

Other than write_custom_geom_only, all parameters are optional (if a user didn't specify these, cryo_fit2 will use default values).

  • For example:

    % phenix.cryo_fit2 model.pdb map.ccp4 3.5 user_custom_geom.eff

Output as a static structure

A final cryo_fitted structure: output/cryo_fit2_fitted.pdb

How to show map contour as mesh

In pymol commandline:

% load ./user_map.ccp4, map1, 1 , ccp4

% isomesh mesh1, map1, 1.5, (all), 0, 1, 3.0

% cmd.color("blue","mesh1")

Otherwise, use pymol GUI:

% A (action) -> Mesh -> @ Level 3.0

How to generate and record movie?

Generate_record_movie_by_cryo_fit2

FAQ

See the cryo_fit2 FAQ

Options

All options will be used as default if unspecified. e.g. secondary_structure_enabled =True

List of most useful options

Option Default value Description of inputs and uses
resolution none A cryo-EM map resolution (angstrom) It needs to be specified by a user.
explore False If True, cryo_fit2 will use maximum number of multiple cores to explore the most optimal MD parameters. However, this exploration requires a lot of computing power (e.g. > 128 GB memory, > 20 cores). Exploring at a macbook pro (16 GB memory, 2 cores) crashed
start_temperature 300 Start temperature (Kelvin) for molecular dynamics simulation
final_temperature 0 Final temperature (Kelvin) for molecular dynamics simulation
MD_in_each_cycle 4 Cycle is each iteration of MD from start_temperature to final_temperature.
number_of_steps 100 Number of MD simulation steps at each temperature
map_weight (automatically optimized) A weight toward cryo-EM map
map_weight_multiply 1 Cryo_fit2 will multiply cryo-EM map weight by this much. Usually when the initial atomistic model has evenly distributed map fitting need, 1 is enough. On the other hand, when the initial atomistic model has un-even distribution of map fitting need, high value (~15) is required.
secondary_structure.en abled True Most MD simulations tend to break secondary structure. Therefore, turning on this option is recommended. If HELIX/SHEET records are present in supplied .pdb file, automatic search of the existing secondary structures in the given input pdb file will not be executed.
secondary_structure.pr otein.remove_outliers True False may be useful for very poor low-resolution structures by ignoring some hydrogen "bond" if it exceed certain distance threshold
output_dir output Output folder name prefix
keep_progress_on_screen True If True, temp=xx dist_moved=xx angles=xx bonds=xx is shown on screen rather than cryo_fit2.log
keep_origin True If True, write out model with origin in original location. If False, shift map origin to (0,0,0)